PROJECT SUMMARY Antiviral cytotoxic CD8+ T lymphocytes (CTLs) are critical for controlling viremia and are central to HIV cure/remission strategies. However, there is a fundamental gap in understanding the molecular determinants underlying the onset and persistence of HIV-specific CTL dysfunction in individuals who fail to control viremia, preventing strategies to restore CTL function for HIV prevention and durable remission. The objective of this application is to define molecular regulation and biomarkers of HIV-specific CTL dysfunction. Based upon strong preliminary evidence, we hypothesize that initiation of CTL dysfunction preceding loss of virologic control and maintenance of CTL dysfunction during antiretroviral therapy are mediated by cell-intrinsic mechanisms, identification of which will inform therapeutic interventions to combat HIV persistence. We will define regulatory mechanisms of CTL dysfunction using an integrative, multi-omics systems biology approach at intra-patient, antigen-specific, and single-cell resolution, and validate using gene editing for restoration of antiviral CTL function. The rationale of the proposed studies is that a more precise understanding of the molecular mechanisms governing durable versus failed CTL-mediated HIV control will be needed to advance CTL-based HIV cure strategies. Aim 1 will define mechanisms by which CTL dysfunction is initiated preceding breakthrough viremia in HIV controllers. Preliminary data demonstrate that a progressive loss of antiviral CTL function precedes viral rebound in patients who lose HIV control. Experiments in this aim will elucidate regulatory pathways governing the initiation of CTL dysfunction by evaluating changes in epigenetic, transcriptional, and post-transcriptional signatures using longitudinal specimens preceding loss of HIV control. Regulatory pathways will be disrupted using gene editing for mechanistic validation. Aim 2 will define mechanisms by which CTL dysfunction is maintained during pharmacologic HIV suppression and the extent to which function can be restored. Preliminary evidence indicates that CTL dysfunction is not restored by antiretroviral therapy or immune checkpoint blockade. Experiments in this aim will identify molecular mechanisms by which dysfunction is maintained in non-escaped HIV-specific CTLs during antiretroviral therapy using a multi-omics approach, and determine the extent to which CTL function can be restored by inhibition and gene editing of identified regulatory networks. This approach is innovative because it couples controlled intra-patient comparisons of epitope-specific CTLs by population and single-cell transcriptional, post-transcriptional, and epigenetic profiling with gene editing in primary cells to interrogate the molecular mechanisms that underlie HIV-specific CTL dysfunction. The proposed research is significant because it will provide the mechanistic groundwork for development and evaluation of interventions that aim to harness CTLs for eradication or functional cure of HIV infection.